The overall goal of this renewal research proposal is to determine the role of ischemia in the development of detrusor overactivity in the non-obstructed bladder. Another goal is to examine the efficacy of targeting cellular stress response pathways to prevent or reverse bladder dysfunction. Lower urinary tract symptoms (LUTS) are bothersome constellation of voiding symptoms with significant impact on quality of life. The prevalence of bladder dysfunction and LUTS among Veterans is almost five folds higher in comparison with its incidence in the general population. It was shown that Veterans with LUTS had a worse quality of life score than Veterans without LUTS. In most cases, particularly in elderly Veterans, LUTS resulted in insomnia, anxiety, fatigue, and depression. Most cases of LUTS in male are attributed to bladder outlet obstruction (BOO) due to benign prostatic enlargement (BPE). However, it has been shown that in approximately one third to more than one half of cases, LUTS are not associated with BPE or BOO. These observations suggest that, in addition to BOO, aging-related local changes in the bladder contribute to LUTS. Growing evidence from basic and clinical research suggests that aging-associated bladder ischemia play a key role. Impairment of human bladder blood flow and the development of bladder ischemia have been verified in elderly patients with LUTS. However, the underlying mechanisms contributing to detrusor overactivity in bladder ischemia remain largely unknown. Our preliminary data suggest that ischemia provokes cellular stress and compromises cellular defensive capacity by impairing the cellular energy sensor adenosine monophosphate-activated protein kinase alpha-2 (AMPK- ?2). Cell stress and defective AMPK-?2 give rise to a unique stress response RNA with two complementary strands namely double-stranded RNA (dsRNA) leading to activation of the AMPK-?2/dsRNA stress response pathway. The AMPK-?2/dsRNA pathway seems to compromise muscarinic M2 and M3 receptors, provoke post-translational modifications of contractile proteins, increase smooth muscle contractions and engender detrusor overactivity. We hypothesize that ?chronic ischemia is an independent factor in the development of detrusor overactivity in the non-obstructed bladder. The mechanism involves activation of cellular stress response via AMPK-?2/dsRNA pathway that triggers overactive bladder contractions by modification of smooth muscle contractile elements?. Using a well-established bladder ischemia model along with knockout mice and cell culture transfection and gene deletion technologies, we propose three specific aims. In aim I, we will define molecular regulation of the AMPK-?2/dsRNA stress response pathway in bladder ischemia. We will determine the mechanism of AMPK-?2 impairment, quantify and clone dsRNA in bladder ischemia and define crosstalk mechanisms between AMPK-?2 and dsRNA. In aim II, we will define regulation of overactive contractions by AMPK-?2/dsRNA pathway in bladder ischemia. We will determine how AMPK-?2/dsRNA pathway provokes overactive bladder contractions by transcriptional and post-translational modifications of muscarinic M2 and M3 receptors. We will examine therapeutic strategies to prevent stress-regulated modifications of M2 and M3 and reverse detrusor overactivity. In aim III, we will define signaling mechanisms downstream of AMPK-?2/dsRNA pathway that modify contractile proteins in bladder ischemia. We will determine the role of AMPK-?2/dsRNA- regulated redox and PKR signaling in modifications of actin-?1 and myosin heavy chain (MHC) and regulation of smooth muscle calcium uptake. We will examine therapeutic strategies to prevent redox- and PKR-mediated actin-?1 and MHC modifications and reverse detrusor overactivity in bladder ischemia. At the conclusion of these studies, we will have: (1) provided new insights into the pathophysiology of detrusor overactivity in the non-obstructed bladder; (2) elucidated the molecular link between AMPK-?2/dsRNA pathway and overactive bladder contractions and 3) defined stress sensitization mechanisms in overactive bladder contractions. Our proposed research may lead to novel diagnostic and therapeutic strategies against detrusor overactivity/LUTS.